One of the major tasks of today's medicine is the development of a therapeutic system which is capable of selectively influencing the expression of a gene suspicious of being misregulated or known to be misregulated. Depending on the gene(s) affected, this may manifest in severe diseases such as e.g. cancer. Although there has been a lot of effort in this field, there is still the need to improve the results gained thus far and combine them e.g. with a system capable of easily monitoring the therapeutic effect and/or the delivery of the therapeutic compound.
In general, the concept of gene expression can be summarized as to include several steps: on the DNA-level, a specific sequence is coding for a protein or an RNA, both of which may exhibit a specific function. In a first step, the DNA is transcribed into corresponding RNA. This step is tightly controlled including so called promoter regions, often at the 5′-end and in close proximity to the coding sequence, as well as further up- or downstream regulatory DNA elements and transcription factors positively and negatively influencing transcription. In case a protein is the final product of the expression of a gene, the transcribed RNA is referred to as mRNA. In a second step, this mRNA is translated into protein and said protein is then in a subsequent step optionally further modified post-translationally. In case an RNA is the final product, the transcribed RNA may be rearranged and processed as well as included in complexes comprising e.g. proteins. Thus, it is possible to influence gene expression on a DNA-level by e.g. targeting factors influencing the first step, namely transcription. Also, the second step may be targeted: in case of a protein as final product, translation of the mRNA, in case of an RNA as final product, the processing etc. may be blocked. If a method ultimately results in the degradation of the transcribed RNA, no final product can be obtained and thus the second step is inhibited.
In case a disease is caused by downregulation of a gene or even by its complete silencing, the therapeutic effect should be the upregulation of said endogenous gene to a normal level. For certain types of cancer, for example, prominent tumour-suppressor genes are known to be silenced and thus unable to exhibit their corresponding tumour-suppressor function. DNA-methylation has been identified as a major reason for gene-downregulation/silencing. Said methylation is often found in the promoter regions of the corresponding genes. In these cases, gene expression may be restored by removing methyl groups from the DNA. Downregulation/silencing of a gene may also be due to the upregulation of a transcription factor which is acting as negative transcription factor for this gene, thus blocking the expression of the gene. In these cases, therapy should be directed to a downregulation of the gene encoding the transcription factor and thus lowering the level of the transcription factor.
If upregulation of a gene is causative for a disease, the therapeutic effect should correspondingly be the downregulation of said endogenous gene to a normal level. In almost all types of cancers, genes are known to be upregulated. They are commonly referred to as oncogenes or proto-oncogenes wherein proto-oncogenes may not directly have an effect on e.g. the proliferation of a cell but indirectly by e.g. causing other genes to be upregulated. RNAi-methods may be used in order to downregulate gene-expression. In contrast to demethylating drugs which are directly acting on the “DNA-level”, RNAi-methods typically act on the second step of gene expression, namely on the translation into protein or the processing into corresponding RNA. These methods ultimately lead to the degradation of the transcribed RNA. Thus, the entire process of gene expression is blocked by RNAi. This may also be referred to as “silencing” of a gene by RNAi. Mechanistically, the artificially introduced “RNA” (e.g. transcribed from a vector, introduced as RNA-duplex, etc.) is processed, and one strand is incorporated into a complex which recognizes transcribed RNA in a sequence specific manner. Thus, the sequence of the introduced RNA specifies the targeted “gene to be silenced”. Following hybridization, recognition and complex formation, the targeted RNA is degraded by further mechanisms.
Current methods of monitoring the effect of therapeutic systems influencing the expression of genes mainly rely on comparing the status of a disease, e.g. a tumour, before and after the therapeutic system is applied. Thus, e.g. X-ray- or ultrasound-methods may be used to determine the size of a tumour prior to and following the treatment. However, with such systems it is neither possible to directly monitor the effect of said treatment on the gene expression nor to analyze whether the corresponding compound localized to the affected tissue at all. Other methods require tissue-samples in order to determine the result of a treatment. Thus, a biopsy or even surgical steps are necessary, which are often accompanied by additional problems such as infections. Furthermore, again only an ex post analysis is possible.
As a consequence, there is a need to establish or improve therapies influencing gene expression and methods that allow monitoring the application and the effect of these therapies on the expression of targeted genes in a non-invasive manner.